Kidney stone growth through the lens of Raman mapping

Bulk composition of kidney stones, often analyzed with infrared spectroscopy, plays an essential role in determining the course of treatment for kidney stone disease. Though bulk analysis of kidney stones can hint at the general causes of stone formation, it is necessary to understand kidney stone microstructure to further advance potential treatments that rely on in vivo dissolution of stones rather than surgery. The utility of Raman microscopy is demonstrated for the purpose of studying kidney stone microstructure with chemical maps at ≤ 1 µm scales collected for calcium oxalate, calcium phosphate, uric acid, and struvite stones. Observed microstructures are discussed with respect to kidney stone growth and dissolution with emphasis placed on < 5 µm features that would be difficult to identify using alternative techniques including micro computed tomography. These features include thin concentric rings of calcium oxalate monohydrate within uric acid stones and increased frequency of calcium oxalate crystals within regions of elongated crystal growth in a brushite stone. We relate these observations to potential concerns of clinical significance including dissolution of uric acid by raising urine pH and the higher rates of brushite stone recurrence compared to other non-infectious kidney stones.


Mixed-phase false-color maps
False-color maps of mixed COD, COM, HAp, calcite, and cyanoacrylate were constructed using the Mapping Review function of Renishaw's WiRE software (v5.6).The non-negative least squares fit produces a Map value for each component with higher numbers indicating better fit.For each component, pixels below its ≈20 th percentile map value were set to be transparent.The multicomponent maps were constructed by layering each component on a black background.For Figure 5, the layer order is calcite (bottom), hydroxyapatite, cyanoacrylate, COM, and COD (top).This means, or areas of mixed COD/COM, that COD will cover COM and often appears as a darker yellow as map value of COD decreases as the map value for COM increases.
For Figure 6, the layer order is COM (bottom), cyanoacrylate, and COD (top).
Figure S3: Raman spectrum of uric acid powder from 400-1800 cm -1 using 785 nm laser.the oriented COM spectrum (green) in the top plot, the fraction of COD present was estimated to be 13% when least-squares fitting with separate spectra for COM as in Figure S8.When using Raman spectra from powdered COD and COM, COD fraction was 42%.All spectra used for the least-squares fit were normalized to a mean variance of 1. COD appears to be identified due overlap between the COM peaks at 1464 and 1490 cm -1 .However, in most spectra identified as COD its minor peaks at 913 and 509 cm -1 not present while minor peaks for COM at 897 and 864 cm -1 are visible when the carboxylate stretch at 1490 cm -1 dominates making it likely that only COM is present.

Figures
Figures S1-S7: Raman spectra for each powdered mineral S4-S8 Figure S8: COM spectra with either the 1464 cm -1 and 1490 cm -1 peak dominant S9 Figure S9: COM stone fit with Raman spectra of powdered minerals vs. oriented COM S10 Figure S10: White light images for brushite stone in Figure 4. S11-S12 Figure S11: White light image for Figure 5 S13 Figure S12: Individual component maps of Figure 5 S13 Figure S13: Comparison of calcium carbonate spectra S14 Figure S14: SEM image of hexagonal COM plates growing on COD S15 Figure S15: Spectra comparison for dark and light regions of crystal in Figure 5, region C S16 Figure S16: White light image for map in Figure 6 S17

Figure S7 :
FigureS7: Raman spectrum of cyanoacrylate from 400-1800 cm -1 using 532 nm laser.Cyanoacrylate was clear and had cured at room temperature for 1 week.

Figure S8 :
Figure S8: Raman spectrum of COM crystal in different orientations.

Figure S9 :
Figure S9: Top: overlayed spectra of COM powder, COD powder, and oriented COM spectrum from Figure 2.Bottom images: 2b, 2d, and 2f as in Figure 2. COM powder and COD powder: least-squares fit map using Raman spectra from powdered minerals.

Figure S10a :
Figure S10a: White light image for brushite stone in Figure 4a.Box indicates area mapped.

Figure S11 :
Figure S11: White light image of area mapped in Figure 5. Earlier laser damage is indicated by blackened areas where protein was carbonized.

Figure S12 :
Figure S12: Individual component maps used to construct Figure 5 (excluding cyanoacrylate).Pure black (RGB values of 0,0,0) areas were made transparent for all but the bottom layer (calcite) to achieve overlay effect.

Figure S13 :
FigureS13: Comparison of calcium carbonate from an initial survey run (green) and high-resolution map (orange) compared to calcite from the RRUFF database (blue) for sample shown in Figure5and FigureS12.Presence of apatite is still apparent in both kidney stone spectra.In the initial survey run, CaCO3 appears to be amorphous or mixed calcite/amorphous as peaks are shifted to lower energy and broadened compared to pure calcite. 1 After polishing and/or time, the spectrum matches that of calcite.Calcite can be distinguished from aragonite by the presence of the peak at 280 cm -1 .

Figure S14 :
Figure S14: Scanning electron microscopy image of stacked hexagonal plates of calcium oxalate monohydrate growing on a calcium oxalate dihydrate blade.

Figure S15 :
Figure S15: COM spectra of unmasked regions (grey areas) in COM crystal in Figure 5, Region C. Autofluorescence is higher in darker regions of this crystal, potentially indicating increased concentration of organic material.

Table S1 :
Raman collection settings for each map presented.